Tuberculosis (TB) remains a significant international public health threat, particularly for US military personnel who are often deployed to areas of high TB prevalence. Mycobacterium tuberculosis (Mtb) is a respiratory pathogen spread via inhalation of infectious airborne particles. Most infected individuals develop protective immunity that serves to contain the organism, but approximately 10% will eventually develop active TB. The localization of mycobacteria-specific CD4+ T cells to the lung appears to be critical to protection against Mtb infection, and may not be optimized by current TB vaccination with intradermal (ID) M. bovis BCG. Human studies using lung cells obtainable by bronchoalveolar lavage (BAL) provide a means to assess local immune responses to Mtb that may be uniquely relevant to evaluating novel TB vaccines. The distinct nature of local immunity within the lung has been further emphasized by recent murine studies demonstrating that respiratory infection is followed by the development of CD4+ memory T cells that are localized to the lung parenchyma and do not rejoin the general circulation. These tissue-resident memory T cells (TRM) display a distinct phenotype, and also show increased capacity to protect against respiratory infection with Mtb. The use of intravenous (IV) injection of pan-leukocyte antibodies to identify T cells that are not in communication with the vasculature has provided a means to sort pulmonary TRM from vascular-associated memory cells. This intriguing approach has not yet been applied to clarifying the significance of BAL-based studies of immunity to Mtb; this step is critical, however, to the ultimate application of these insights to human studies. The overall goal of the current proposal is to clarify the mechanisms by which CD4+ T cells within BAL differ from and interact with other lung CD4+ T-cell populations to mediate lymphocyte recruitment to the lung and, ultimately, protection against respiratory challenge with Mtb. Our research team is uniquely qualified to address these issues, as it includes investigators with experience in bronchoscopy-based studies of human immunity to Mtb (Richard Silver, PI), murine assessments of immunity to Mtb (W. Henry Boom, Consultant) and optimization of immune assays involving lung cells from both mice and humans (Tracey Bonfield, Co-investigator). We will also greatly benefit from the involvement of a pioneer in the application of TRM methodology to the study of Mtb infection (Daniel Barber of NIAID, Consultant). Our studies will utilize a murine model of Mtb infection in which lung homogenate cells stained by IV injection (?IV+ T cells?) associated with the lung vasculature are sorted from T cells that cannot be labeled in this manner. These ?IV- T cells? predominantly display a TRM phenotype and are retained within the parenchyma. We will apply this approach to evaluate the interactions of IV- and IV+ lung CD4+ T cells and to clarify their relationship to BAL CD4+ T cells in mice. Parallel human studies will utilize both baseline BAL cells and unique samples obtained by modeling recall responses to Mtb protein antigens using bronchoscopic segmental antigen challenge with purified protein derivative of Mtb (PPD). These approaches will be integrated to address the following Specific Aims: 1) To determine the mechanisms by which mycobacteria-specific CD4+ T cells in BAL are phenotypically distinct from IV- and IV+ lung homogenate CD4+ T-cell populations; 2) To determine the mechanisms by which BAL CD4+ T-cells interact with IV- and IV+ CD4+ lung T-cell populations to recruit additional T cells to the lung parenchyma and airways; 3) To determine the mechanisms by which BAL and lung parenchymal CD4+ T cells interact to mediate protection against respiratory infection with virulent M. tuberculosis.